The development of therapies to prevent preterm birth, which is the major cause of neonatal morbidity and mortality, requires a clear understanding of the hormonal interactions that transform the myometrium from the relaxed to the laboring state. In this context, progesterone is a major player because it promotes myometrial relaxation for most of pregnancy, its withdrawal initiates labor, and progestin prophylaxis decreases the incidence of preterm birth in a subset of women. The proposed research examines how progesterone exerts these actions by determining how, via its interaction with its receptors, PR-A and PR-B, it affects gene expression in human myometrial cells. Our published data suggest that progesterone promotes myometrial relaxation via PR-B and that labor is initiated by increased myometrial cell expression of PR-A, which causes functional progesterone withdrawal by inhibiting the transcriptional activity of PR-B. To test this hypothesis we developed a human myometrial cell line to examine how PR-A and PR-B affect genomic actions of progesterone. We found that progesterone was anti-inflammatory and inhibited the expression of pro- inflammatory genes in PR-B-dominant cells but had the opposite effect in PR-A-dominant cells. The effects appeared to be mediated by direct actions of the PRs at the promoters of pro-inflammatory genes and by modulation of nuclear factor -B (NFB) activity; especially by increased expression of IB, a major repressor if NFB. These findings are important because labor is thought to be sequelae of myometrial inflammation. Thus we propose that progesterone via PR-B promotes myometrial relaxation by inhibiting NF-B-mediated pro-inflammatory gene expression, whereas it promotes labor via PR-A-mediated up-regulation of pro- inflammatory gene expression. To test this hypothesis we will: 1) determine how progesterone via its interaction with PR-A and PR-B affects basal and cytokine-induced expression of prostaglandin (PG) - endoperoxide synthase 2 (PTGS2; chosen as a prototypical pro-labor/pro-inflammatory gene) and IB in human myometrial cells (Specific Aim 1). We will characterize the mechanism by which PR-A and PR-B interact at the PTGS2 and IB promoters, and how they may modulate NF-B activity. Another important concept to arise from our research is that the parturition-associated increase in myometrial cell PR-A expression, which is a key event in human parturition, is up-regulated by PGF2. Specific Aim 2 wil be to identify the intracellular signaling pathways and cis/trans regulators through which PGF2 affects PR-A expression in human myometrial cells. The signaling pathways through which PGF2 via its receptor affects PR-A expression and the cis elements in the PR-A promoter and the transcription factors acting on those elements through which PGF2 induces PR-A expression will be identified. Experiments for both aims will be performed in human myometrial cell lines, including our line that expresses PR-A and PR-B in response to independent inducers, and myometrial explant and primary myocyte cultures of term myometrium. The research will advance understanding of how progesterone controls human pregnancy and parturition and provide knowledge needed to develop/improve progestin-based therapies to prevent preterm birth. PUBLIC HEALTH RELEVANCE: Preterm birth is a major socioeconomic problem that affects 10-15% of pregnancies and causes 70-80% of neonatal mortality and morbidity. Despite advances in neonatal care that have improved survival outcomes, the confounding problems due to preterm birth profoundly affect a preterm infant's future health. Clearly, the final phase o fetal development is best achieved in the womb environment rather than in the neonatal intensive care unit. To this end, we must prevent and/or suppress preterm labor. However, current therapies to suppress preterm labor are generally ineffective. To solve this problem we must fill the knowledge-gaps that limit our capacity to develop effective treatments for preterm labor. To this end, our goal is to elucidate the hormonal interactions that control uterine contractility during human pregnancy. In this context the steroid hormone progesterone is critical. This proposal addresses the molecular mechanisms by which the nuclear progesterone receptors PR-A and PR-B mediate progesterone actions in human myometrial cells. A clear understanding of this process may reveal novel therapeutic targets for the suppression of preterm labor and the prevention of preterm birth.